Hyperoxia is frequently used in the treatment of pulmonary insufficiency in premature infants and adults with acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS). However, in infants, hyperoxia contributes to the development of Broncho pulmonary dysplasia (BPD) in infants, and exacerbates lung injury in ALI/ARDS patients. Studies from our laboratory have clearly shown a mechanistic role for cytochrome P450 (CYP)1A enzymes in oxygen injury. The central hypothesis of this research is that hyperoxia induces pulmonary cytochrome P450 (CYP)1A enzymes in vivo by forming novel endogenous Ah receptor (AHR) ligands, and that hepatic CYP1A2 protects against hyperoxic lung injury by metabolizing reactive oxygen species (ROS)-mediated molecules and/or linoleic acid-derived leukotoxin epoxides or diols) that cause lung injury. The hypothesis that specific ROS-mediated oxidative DNA lesions in humans could serve as novel biomarkers of human ALI/ARDS will also be tested. In order to test the above-mentioned hypotheses, we propose the following Specific Aims: 1. To test the hypothesis that 6-formyllindolo[3,2-b]carbazole (FICZ) is the novel endogenous ligand of the Ah receptor (AHR) that induces hepatic and pulmonary CYP1A enzymes in vivo and in vitro under hyperoxic conditions. This aim has two sub-aims: To elucidate the role of FICZ and/or AHR in the induction of human CYP1A by hyperoxia in vivo. (ii). To unravel the molecular mechanisms of CYP1A1 induction in human lung cells by hyperoxia, and test the hypothesis that FICZ (formed under hyperoxic conditions) contributes mechanistically to CYP1A1 induction. 2. To determine the molecular mechanisms by which liver CYP1A enzymes contribute to hyperoxic lung injury. We hypothesize that leukotoxin epoxides and diols will accumulate in lungs of Cyp1a2-null mice, and will contribute to the increased susceptibility to hyperoxic lung injury. We will create a humanized knock-in mouse model, which will express human CYP1A2 in a liver-specific manner (using Crealb transgenic mice driven by albumin promoter) in mice on a Cyp1a2-null background. We will determine if these mice will be rescued against hyperoxic lung injury. 3. To determine the mechanistic role of CYP1A enzymes in oxidative DNA damage-mediated by hyperoxia. This aim has two sub-aims: (i) to test the hypothesis that mice lacking the genes for CYP1A1 and or 1A2 are more susceptible to pulmonary oxidative DNA damage upon hyperoxic exposures than WT mice, and that novel oxidative DNA lesion mechanistically contribute to hyperoxic lung injury in mice and ARDS in humans. (ii) To test the hypothesis that endotracheal aspirates of ALI/ARDS patients will display specific oxidative DNA lesions that could serve as novel biomarkers for ALI/ARDS. Successful accomplishment of the aims could lead to the development of novel biomarkers of ALI/ARDS, and new strategies for the prevention and/or treatment of ALI/ARDS in humans.